CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb, Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC acid MCC, we wished to determine if this was also the case in MCC, Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and I on 9p. No loss of heterozygosity (LO H) was peen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p, Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined, Half of all informative cases had LOH at D95168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168, A second region (InFNA-D9S126) showed L0H in 10(44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all Il tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p 14' antibody, These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus. (C) 2001 Wiley-Liss, Inc.

Efficacy of praziquantel against Schistosoma japonicum: field evaluation in an area with repeated chemotherapy compared with a newly identified endemic focus in Hunan, China

This study conducted in 1999/2000 was designed to evaluate the efficacy of praziquantel against Schistosoma japonicum in an area with repeated chemotherapy (Area A) compared with a newly identified endemic focus (Area B) in Hunan Province, China. The population size was 2015 and 2180 in Areas A and B, respectively, of which 1129 and 1298 subjects received stool examination. A total of 230 subjects were identified by the Kato-Katz technique (4 smears per person) as being infected with S. japonicum, 124 in Area A (prevalence 11 %) and 106 in Area B (prevalence 8.2%). They were treated with a single oral dose of praziquantel (40 mg/kg) in the non-transmission season. A follow-up stool examination was made 50 days after treatment. Among the 220 cases followed, 22 were found stool-egg-positive, with an overall cure rate of 90 %, and 99 % reduction of infection intensity (eggs per gram stool). No significant difference was found in cure rates between the 2 areas (89.7% vs 90.3%). The efficacy of the drug in the area with repeated chemotherapy was not significantly different from that in the newly identified endemic focus. This study, therefore, suggests that the efficacy of praziquantel against S. japonicum has not changed in the Dongting Lake region after more than 14 years of mass chemotherapy, and there is no evidence of tolerance or resistance of S. japonicum against praziquantel.

Compartmentalization of Ras proteins

The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and :K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effecters and exchange factors.

GTP-dependent segregation of H-ras from lipid rafts is required for biological activity

Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.

Evidence for three tumor suppressor loci on chromosome 9p involved in melanoma development

Cytogenetic and loss of heterozygosity (LOH) studies have long indicated the presence of a tumor suppressor gene (TSG) on 90 involved in the development of melanoma, Although LOH at 90 has been reported in approximately 60% of melanoma tumors, only 5-10% of these tumors have been shown to carry CDKN2A mutations, raising the possibility that another TSG involved in melanoma maps to chromosome 90. To investigate this possibility, a panel of 37 melanomas derived from 35 individuals was analyzed for CDKN2A mutations hy single-strand conformation polymorphism analysis and sequencing. The melanoma samples were then typed for 15 markers that map to 9p13-24 to investigate LOH trends in this region. In those tumors demonstrating retention of heterozygosity at markers flanking CDKN2A and LOH on one or both sides of the gene, multiplex microsatellite PCR was performed to rule out homozygous deletion of the region encompassing CDKN2A. CDKN2A mutations were found in tumors from 5 patients [5 (14%) of 35], 4 of which demonstrated LOH across the entire region examined. The remaining tumor with no observed LOH carried two point mutations, one on each allele, Although LOH was identified at one or more markers in 22 (59%) of 37 melanoma tumors corresponding to 20 (57%) of 35 individuals, only 11 tumors from 9 individuals [9 (26%) of 35] demonstrated LOH at D9S942 and D9S1748, the markers closest to CDKN2A. Of the remaining 11 tumors with LOH, 9 demonstrated LOH at two or more contiguous markers either centromeric and/or telomeric to CDKN2A while retaining heterozygosity at several markers adjacent to CDKN2A. Multiplex PCR revealed one tumor carried a homozygous deletion extending from D9S1748 to the IFN-alpha locus. In the remaining eight tumors, multiplex PCR demonstrated that the observed heterozygosity was not attributable to homozygous deletion and stromal contamination at D9S1748, D9S942, or D9S974, as measured by comparative amplification strengths, which indicates that retention of heterozygosity with flanking LOH does not always indicate a homozygous deletion, This report supports the conclusions of previous studies that at least two TSGs involved in melanoma development in addition to CDKN2A may reside on chromosome 9p.

No evidence of a role for activating CDK2 mutations in melanoma

Inactivation of p16(INK4a) and/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. Activating CDK4 mutations prevent the binding and inhibition of CDK4 by p16(INK4a). A second, more indirect role for CDK4 is in late G(1), where It may sequester the inhibitors p27(KIP1) or p21(CIP1) away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G(1) into S phase. As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27(KIP1) or p21(CIP1). However, except for a silent polymorphism, we detected no variants within this region of the CDK2 gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of the CDK2 gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigen pmel17, which maps less than 1 kb away in head to head orientation with CDK2 and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated. (C) 2001 Lippincott Williams & Wilkins.

The impact of the Human Genome Project on medical genetics

The near completion of the Human Genome Project stands as a remarkable achievement, with enormous implications for both science and society. For scientists, it is the first step in a complex process that will lead to important advances in the diagnosis and treatment of many diseases. Society, meanwhile, must prevent genetic discrimination, and protect genetic privacy through appropriate legislation.

Adoptive transfer of autologous Epstein-Barr virus-specific cytotoxic T cells for nasopharyngeal carcinoma

Tumor cells from NPC patients are regularly and latently infected with EBV. To examine whether the virus serves as target for immune intervention of the cancer, we determined levels of EBV-specific CTLp in peripheral blood from NPC patients, long-term survivors of the cancer and healthy subjects. CTLp levels of test subjects varied between 3-3,000/10(6) PBMCs. The plasma EBV burden increased when the CTLp level fell below 150, whereas the EBV burden of PBMCs was not correlated with CTLp level. Compared with healthy carriers, CTLp levels of patients were lower and varied over a wider range, between 3-1,500/10(6) PBMCs. The quantitative immune deficit was probably attributed to the tumor because, first, CTLp in survivors was restored to levels similar to those in healthy carriers after the tumor had been successfully treated. Second, the CTLp level changed as disease progressed, being lower in local disease, increased in locoregional disease and decreased again when the tumor metastasized. Based on these findings, 4 patients with advanced disease were infused with 5 x 10(7)-3 x 10(8) autologous EBV CTLs. The treatment was safe and unaccompanied by inflammatory or other complications, but whether it improved tumor control could not be discerned from the large tumor bulk. Nevertheless, the treatment regularly increased CTLp levels of patients, maintained it at higher levels for protracted periods and, in 3 patients, restored host surveillance of EBV replication, reducing the plasma EBV burden. Taken together, these results provided a rationale to further explore EBV as a target of immune intervention of NPC. (C) 2001 Wiley-Liss, Inc.